Bringing home the carbon: photorespiratory CO2 recovery shows diverse efficiency in Brassicaceae

This article comments on: Schlüter U, Bouvier JW, Guerreiro R, Malisic M, Kontny C, Westhoff P, Stich B, Weber APM. 2023. Brassicaceae display variation in efficiency of photorespiratory carbon-recapturing mechanisms. Journal of Experimental Botany 74, 6631–6649.

Photorespiration has long been regarded as a wasteful pathway, expending both carbon and energy.Yet the process is beginning to emerge as an ambiguous pathway, with our understanding not as clear as previously thought.Studies have shown that establishing a photorespiratory pump from the ancestral C 3 state to reclaim lost CO 2 has been an integral step towards the evolution of C 4 photosynthesis.However, not all plant families contain C 4 relatives, raising interesting questions about why C 4 photosynthesis evolved in some families but not others, and why some species have photosynthetic activity that is neither C 3 nor C 4 and appears to reiterate intermediate stages that occurred during the evolution of C 4 photosynthesis in other families.These plants, termed C 3 -C 4 intermediates or C 2 species, rely on a photorespiratory shuttle to concentrate carbon.Here, Schlüter et al. (2023) compile a convincing case for metabolic diversity in the C 3 -C 4 intermediates and demonstrate convergent evolution within Brassicaceae combining anatomical, biochemical, physiological, and genotypic evidence to improve understanding of the C 3 -C 4 phenotype.
Photorespiration is the second greatest metabolic flux in the plant, following photosynthesis, occurring when Rubisco fixes O 2 instead of CO 2 , and is more prevalent under hot, dry conditions when stomata close and limit CO 2 uptake into the leaf.The oxygenation reaction can account for up to 25% of Rubisco activity, producing the toxic metabolite 2-phosphoglycolate (2PG) which the plant must detoxify using the energy-demanding photorespiratory pathway, subsequently releasing both CO 2 and NH 3 in the process (Sharkey, 1988).Previous thinking suggested that photorespiration ran as a closed cycle, yet recent studies indicate that the pathway may partly operate in a non-cyclic manner, interacting with other biochemical pathways to balance cellular stoichiometry in accordance with environmental conditions (Timm et al., 2012;Hodges et al., 2016;Busch, 2020).Consequently, this photorespiratory plasticity may allow variation of biochemical phenotypes both within and between species.In addition, it is now known that photorespiration is not the wasteful pathway once thought, with C 3 species, including crops such as rice and barley, reclaiming varying degrees of carbon to recoup expenditure (Busch et al., 2018).Photorespiration occurs mainly in C 3 and C 3 -C 4 intermediate species.The pathway is largely, but not completely, suppressed by the carbon-concentrating mechanism (CCM) that operates in C 4 species, thereby favouring carboxylation by Rubisco over oxygenation (Zelitch et al., 2009).It is thought that photorespiration played an intrinsic role in C 4 evolution, when low atmospheric CO 2 concentrations 25-30 million years ago and warm environments increased flux through the pathway (Christin et al., 2011;Sage et al., 2018).Under these conditions, acquisition of a photorespiratory pump would improve photosynthetic efficiency and give plants a competitive advantage over those without this CO 2 -concentrating mechanism (Fig. 1).This photorespiratory pump (or C 2 photosynthesis) is found in C 3 -C 4 intermediates and optimizes the pathway by splitting the process across two cell types.In these plants, glycine decarboxylase, the enzyme responsible for photorespiratory CO 2 release, is confined to the bundle sheath cells.This not only increases the path length for photorespiratory CO 2 to diffuse out of the leaf, thereby increasing the chances of the CO 2 being recaptured (Rawsthorne et al., 1988), but also raises the concentration of CO 2 around Rubisco up to 3-fold to favour the carboxylation reaction (Keerberg et al., 2014).Figure 2 describes the cellular location of photorespiration and carboxylation in the differing photosynthetic types.Yet, the influence of photosynthetic type on photorespiratory CO 2 recovery raises many interesting questions regarding diversity between phenotypes which are answered here by Schlüter et al. (2023) through closely examining a range of species in the Brassicaceae, which includes several economically important crop species.

Photosynthetic diversity in Brassicaceae
Despite natural occurrence in warm and water-limited environments, which generally favour C 4 photosynthesis, no members of the Brassicaceae use this mode of photosynthesis (Apel et al., 1997).Nevertheless, several C 3 -C 4 intermediate species in this family have acquired a photorespiratory pump, which is thought to be a pre-requisite for C 4 evolution (Sage et al., 2012).This suggests that there was potential for evolution of C 4 photosynthesis in the Brassicaceae, so the absence of this mode of photosynthesis and persistence of C 3 -C 4 intermediacy in some members of the family raises interesting questions.For example, were there other factors that prevented development of C 4 photosynthesis in the Brassicaceae, or could C 3 -C 4 intermediacy be the optimal photosynthetic type for the natural environments where C 3 -C 4 intermediate species occur (Blätke and Bräutigam, 2019)?It should be noted that C 3 -C 4 intermediacy is remarkably rare; among the 380 000 species of plants, only ~50 C 3 -C 4 intermediate species have been confirmed to date (Sage et al., 2018).Of course, the true number is likely to be much higher, as many species have not yet been examined in detail and assignment of a given species to a photosynthetic type is not trivial as it requires assessment of multiple physiological and anatomical parameters (Lundgren, 2020).Currently, the only C 3 -C 4 intermediate species in commercial production is wild rocket (Diplotaxis tenuifolia), a member of Brassicaceae that includes many important C 3 crop species (Lundgren, 2020).Schlüter et al. (2023) investigate the extent of metabolic variation within Brassicaceae, incorporating 75 parameters implicated in photosynthesis to elucidate the diversity present across this intriguing plant family in comparison with their closest C 4 relative-Gynandropsis gynandra from the Cleomaceae (a sister family of the Brassicaceae in the order Brassicales).In doing so, the authors successfully clarify another case of C 3 -C 4 intermediacy in Hirschfeldia incana (HIR3) within the Brassicaceae.HIR3, an accession of H. incana, is genetically distinct from both HIR1 and NIJ accessions, appearing closer to the Sinapis pubescens lineage as identified by Guerreiro et al. (2023), meaning that independent evolution of the photorespiratory shuttle occurred within this lineage.
Additionally, to ease identification barriers, Schlüter et al. (2023) propose recording assimilation data at low CO 2 as their measurements show significant correlations between the CO 2 compensation point and assimilation rate at 50 ppm CO 2 .Another suggestion by the authors is to use chlorophyll fluorescence measurements of photosynthesis efficiency (F v /F m ) and measurements of stomatal conductance as diagnostic parameters for assignment of a given species to a photosynthetic category following positive correlation between the net assimilation rate and electron transport rate.These where Rubisco also assimilates O 2 in up to 25% of its reactions, and the toxic product of O 2 fixation, 2-phosphoglycolate, must be processed by the photorespiratory cycle (Sharkey, 1988).C 3 -C 4 photosynthesis (or C2) B) exploits photorespiration to realize a simple carbon-concentrating mechanism (CCM) by diffusing glycine to the bundle sheath for decarboxylation (Rawsthorne et al., 1988).Through restricting glycine decarboxylase activity to the bundle sheath, the C 3 -C 4 intermediates force a division of photorespiration, which increases CO 2 concentration around Rubisco, at the expense of releasing NH 3 , which must be reassimilated.Consequently, C 3 -C 4 intermediates can reclaim CO 2 previously released from photorespiration more effectively in comparison with C 3 species.In contrast, for C 4 photosynthesis (C), phosphoenolpyruvate carboxylase (PEPC) is the primary assimilatory enzyme that lacks affinity for O 2 , producing a C 4 organic acid to shuttle carbon into the bundle sheath for decarboxylation and raising the concentration of CO 2 in bundle sheath cells, where Rubisco is located.By favouring the carboxylation reaction of Rubisco, the C 4 CCM largely suppresses the need for photorespiration, increasing the efficiency of CO 2 assimilation at the cost of the additional energy needed to regenerate the initial CO 2 acceptor, phosphoenolpyruvate (PEP).Anatomical and biochemical indicators show differences between the photosynthetic types to confirm their identity (see also Fig. 3).C 3 -C 4 intermediates and C 4 species usually present enlarged bundle sheath cells containing an abundance of organelles that are larger than those seen in C 3 species, although lower numbers of mitochondria are found in the bundle sheath cells of C 4 species (Sage et al., 2014).Created with BioRender.com.
recommendations could reduce time-consuming data collection and allow for quicker confirmation of photosynthetic type for future researchers.
In their investigation, Schlüter et al. (2023) examine anatomical, biochemical, and physiological lines of evidence alongside phylogenetic analyses to determine how these parameters interact and their effect on lineage-specific traits.The authors' results show remarkable diversity across 28 species of Brassicaceae, 14 of which were classified as C 3 -C 4 intermediates.Sequence data from 102 orthogroups revealed the phylogenetic relationship of the species, uncovering examples of convergent evolution across five lineages within the Brassicaceae.
The influence of photosynthetic type was found to affect performance under contrasting environmental conditions, with intermediate species showing strengthened CO 2 assimilation rates under low to subambient CO 2 concentrations, owing to their CCM, in comparison with C 3 species.Although the extent of CO 2 reclamation shows broad variation within a species despite photosynthetic type, previous research in rice shows much intraspecific diversity regarding CO 2 recapture between wild and cultivated species, indicating improved nitrogen use efficiency for those with more successful CO 2 recovery rates (Joo et al., 2022).
The C 4 pathway of photosynthesis involves initial fixation of CO 2 in mesophyll cells by phosphoenolpyruvate carboxylase (PEPC), producing C 4 acids (malate and aspartate) that move into bundle sheath cells, where they are decarboxylated to release CO 2 that is refixed by Rubisco.The residual C 3 moieties are shuttled back to the mesophyll cells in the form of pyruvate, alanine, or phosphoenolpyruvate to sustain further CO 2 fixation by PEPC. Figure 2C represents the C 4 subtype NAD-Me, as used by Gynandropsis gynandra in Schlüter et al. (2023), in comparison to C 3 (Fig. 2A) and C 3 -C 4 intermediate (Fig. 2B) modes of photosynthesis.Additionally, Figure 3 shows biochemical dynamics between organelles within the three photosynthetic types.Metabolite profiles by Borghi et al. (2021) examining differing photosynthetic types in the genus Flaveria spp.suggested that aspartate might also be shuttled between cell types in C 3 -C 4 intermediates.Schlüter et al. (2023) found no evidence of elevated PEPC activity or C 4 characteristics of metabolite shuttling in the Brassicaceae, but did observe other metabolic and anatomical distinctions between photosynthetic types including multiple amendments that improve the photosynthetic efficiency and potential biological fitness of C 3 -C 4 species in specific environments.In particular, D. tenuifolia exhibited a remarkably low CO 2 compensation point, almost in line with values usually representative of C 4 species, meaning that a particularly efficient C 2 mechanism is present in this species.High levels of malate, aspartate, and glutamate were also found in both D. tenuifolia and Moricandia arvensis in line with their low CO 2 compensation points.Analysis of metabolite pools showed much variation within all species, especially for serine levels, in agreement with Fu et al. (2023) who found 23-41% variation of serine export under differing photorespiratory conditions in C 3 species.Under ambient (421 ppm) CO 2 , Schlüter et al. (2023) did not find any advantages for the C 3 -C 4 taxa; however, under ≤200 ppm CO 2 , the intermediate photosynthetic type showed more pronounced water use efficiency than the C 3 species, suggesting a higher tolerance of drought conditions.

Climate resilience: future-proof C 3 -C 4 intermediates
The progression of climate change brings great uncertainty for future food production, alongside an urgent need for solutions to feed an ever-growing global population.Drastic weather fluctuations mean crop species must be resilient to variations in seasonal patterns whilst economizing on resource inputs such as water and nitrogen to improve agricultural sustainability.Future growing seasons are likely to become hotter and drier, on average, and more unpredictable especially in tropical regions, where drought conditions will often be followed by flooding, posing a further risk to crop production.Additionally, by 2050, the atmospheric CO 2 concentration is expected to rise to 550 ppm, meaning a greening of the planet over the coming years as C 3 species flourish under high CO 2 levels owing to a reduction in photorespiratory flux increasing carbon gain (Jaggard et al., 2010).Yet, high CO 2 concentrations can also instigate stomatal closure, again giving rise to photorespiration, leaving the response of C 3 species to climate change difficult to foresee.Observations from free air CO 2 enrichment studies have shown varying effects of elevated CO 2 on different crop species, with positive effects on photosynthetic activity often being offset by disturbance of C/N balance and source-sink relations (Ainsworth and Long, 2021).
Fortunately, the C 3 -C 4 intermediates show unique metabolic plasticity compared with other photosynthetic types, meaning that they can switch between C 3 and C 3 -C 4 photosynthesis depending on environmental conditions.This capacity provides many advantages, as photorespiration is stimulated not only when stomata close, but also under saline soil conditions, useful for land liable to flooding.Schlüter et al. (2023) have shown that C 3 -C 4 intermediates can not only efficiently reclaim photorespiratory CO 2 that would otherwise be lost from the leaf, but can also benefit from improved water use efficiency under low CO 2 conditions that promote photorespiration.Consequently, crop improvement strategies using the C 3 -C 4 photorespiratory shuttle are likely to be more adaptable to climatic variability and tolerant to abiotic stress in comparison with both C 3 and C 4 photosynthetic types.Of course, C 4 crop species remain productive under hot, dry, conditions, showing good water and nitrogen use efficiencies.However, under extreme weather fluctuations, the benefits of C 4 metabolism become less apparent, especially under cooler, cloudier conditions.Fig. 3. Biochemical dynamics between organelles and cellular localization of oxygenation and carboxylation reactions of Rubisco in three modes of photosynthesis, as described by Schlüter et al. (2023).C 3 photosynthesis (A) keeps both photosynthesis and photorespiration contained within the mesophyll.Photosynthesis occurs in the chloroplast, but peroxisomes and mitochondria are also required for photorespiration to take place.Photorespiration is needed to prevent accumulation of the toxic molecule 2-phosphoglycolate (2PG) in both C 3 and C 3 -C 4 metabolism.In C 3 -C 4 photosynthesis (B), the carboxylation reaction occurs in both the mesophyll and bundle sheath chloroplasts.In contrast to C 3 photorespiration, the C 3 -C 4 intermediates diffuse glycine from the mesophyll cell to bundle sheath mitochondria for decarboxylation, thus creating a simple carbon-concentrating mechanism, releasing NH 3 and shuttling serine back to the mesophyll cell to return to the chloroplast.The process still maintains distribution across the three organelles, similar to C 3 species, but divides photorespiration across two cell types.The carboxylation reaction of C 4 photosynthesis (C: NAD-ME For Brassicaceae, their lack of C 4 relatives has resulted in a uniquely proficient version of the photorespiratory shuttle in the C 3 -C 4 intermediates facilitated by an augmented organelle arrangement in the bundle sheath as concluded by Schlüter et al. (2023).This anatomical remodelling could also facilitate a non-cyclic mechanism, providing scope for variation within the photorespiratory pathway through an export valve of glycine and serine interacting with other metabolic processes.Given the large number of existing C 3 crop species within the Brassicaceae, this agronomically important family provides ideal candidates for future-proofing food production, both as candidates for domestication and as models for engineering the C 3 -C 4 mechanism into key C 3 species to deliver environmental versatility.Schlüter et al. (2023) demonstrate not only the broad metabolic diversity present within Brassicaceae, but also the means to efficiently identify previously unknown C 3 -C 4 intermediates in undervalued crop species capable of climate resilience.

Fig. 1 .
Fig. 1.Evolution of C 4 photosynthesis from the ancestral C 3 state.C 3 -C 4 intermediates use a photorespiratory CO 2 pump (or shuttle), also termed 'C 2 photosynthesis', referring to the two-carbon molecule 2-phosphoglycolate (2PG) produced from O 2 fixation by Rubisco.2PG is a toxic molecule that must be processed by photorespiration.Some species remain in a stable C 3 -C 4 state without progressing further towards C 4 photosynthesis.Consequently, these species may develop an optimized version of C 2 photosynthesis, becoming a 'super C 2 ' in comparison with species that conform to the evolutionary trajectory from C 3 to C 4 .Each step along the evolutionary continuum requires an orchestration of anatomical and biochemical upgrades to support transitional states.Through identifying these metabolic, anatomical, and physiological markers, the photosynthetic type can be determined.BS, bundle sheath; M, mesophyll; GDC, glycine decarboxylase; PEPC, phosphoenolpyruvate carboxylase.Adapted fromSage et al. (2014).

Fig. 2 .
Fig. 2. Cellular distribution and anatomical differences of the carboxylation and oxygenation of Rubisco in C 3 , C 3 -C 4 intermediate, and C 4 species.In all three photosynthetic types, Rubisco fixes CO 2 to ultimately enter the Calvin-Benson-Bassham cycle.Most plants use C 3 photosynthesis (A),where Rubisco also assimilates O 2 in up to 25% of its reactions, and the toxic product of O 2 fixation, 2-phosphoglycolate, must be processed by the photorespiratory cycle(Sharkey, 1988).C 3 -C 4 photosynthesis (or C2) B) exploits photorespiration to realize a simple carbon-concentrating mechanism (CCM) by diffusing glycine to the bundle sheath for decarboxylation(Rawsthorne et al., 1988).Through restricting glycine decarboxylase activity to the bundle sheath, the C 3 -C 4 intermediates force a division of photorespiration, which increases CO 2 concentration around Rubisco, at the expense of releasing NH 3 , which must be reassimilated.Consequently, C 3 -C 4 intermediates can reclaim CO 2 previously released from photorespiration more effectively in comparison with C 3 species.In contrast, for C 4 photosynthesis (C), phosphoenolpyruvate carboxylase (PEPC) is the primary assimilatory enzyme that lacks affinity for O 2 , producing a C 4 organic acid to shuttle carbon into the bundle sheath for decarboxylation and raising the concentration of CO 2 in bundle sheath cells, where Rubisco is located.By favouring the carboxylation reaction of Rubisco, the C 4 CCM largely suppresses the need for photorespiration, increasing the efficiency of CO 2 assimilation at the cost of the additional energy needed to regenerate the initial CO 2 acceptor, phosphoenolpyruvate (PEP).Anatomical and biochemical indicators show differences between the photosynthetic types to confirm their identity (see also Fig.3).C 3 -C 4 intermediates and C 4 species usually present enlarged bundle sheath cells containing an abundance of organelles that are larger than those seen in C 3 species, although lower numbers of mitochondria are found in the bundle sheath cells of C 4 species(Sage et al., 2014).Created with BioRender.com.